Quinazolin-4 (3h)- one derivatives used as p13 kinase inhibitors

ABSTRACT

The present invention relates to the compound of formula (I) 
     
       
         
         
             
             
         
       
     
     and to compositions comprising the same and to the use of the compound and to compositions of the compound in treatment, for example in the treatment of inflammatory diseases, in particular respiratory inflammatory disease. The invention also extends to methods of making the said compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International PatentApplication No. PCT/GB2011/052015, filed Oct. 18, 2011, which claimspriority to EPO Patent Application No. 099135360 filed Oct. 18, 2010,and PCT International Patent Application No. PCT/EP2010/065746, FiledOct. 19, 2010, the contents of each of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to a compound that inhibits phosphoinositide3-kinases, (PI3 kinases). In particular the invention relates to acompound that inhibits the PI3 kinase delta sub-type and, in addition,the gamma and alpha sub-types thereof, and to its use in therapy,including in pharmaceutical combinations, especially in the treatment ofinflammatory diseases, including inflammatory diseases of the lung, suchas COPD and asthma. The disclosure also extends to methods of preparingthe said compound and pharmaceutical compositions comprising same.

BACKGROUND OF THE INVENTION

Lipid kinases catalyse the phosphorylation of lipids to produce speciesinvolved in the regulation of a wide range of physiological processes,including cellular migration and adhesion. The PI3-kinases are membraneassociated proteins and belong to this class of enzymes which catalysethe phosphorylation of lipids which are themselves associated with cellmembranes. The PI3-kinase delta (δ) isozyme (PI3 kinase δ) is one offour isoforms of type I PI3 kinases responsible for generating various3′-phosphorylated phosphoinositides, that mediate cellular signallingand have been implicated in a number of cellular processes such asinflammation, growth factor signalling, malignant transformation andimmunity [See Review by Rameh, L. E. and Cantley, L. C. J. Biol. Chem.,1999, 274:8347-8350].

Involvement of PI3 kinases in controlling inflammation has beenconfirmed in several models using pan-PI3 kinase inhibitors, such asLY-294002 and wortmannin [Ito, K. et al., J Pharmacol. Exp. Ther., 2007,321:1-8]. Recent studies have been conducted using either selective PI3kinase inhibitors or in knock-out mice lacking a specific enzymeisoform. These studies have demonstrated the role of pathways controlledby PI3 kinase enzymes in inflammation. The PI3 kinase δ selectiveinhibitor IC-87114 was found to inhibit airways hyper-responsiveness,IgE release, pro-inflammatory cytokine expression, inflammatory cellaccumulation into the lung and vascular permeability inovalbumin-sensitized, ovalbumin-challenged mice [Lee, K. S. et al., J.Allergy Clin. Immunol., 2006, 118:403-409 and Lee, K. S. et al., FASEBJ., 2006, 20:455-65]. In addition, IC-87114 lowered neutrophilaccumulation in the lungs of mice and neutrophil function, stimulated byTNFα [Sadhu, C. et al., Biochem. Biophys. Res. Commun., 2003,308:764-9]. The PI3 kinase δ isoform is activated by insulin and othergrowth factors, as well as by G-protein coupled protein signaling andinflammatory cytokines. Recently the PI3 kinase dual δ/γ inhibitorTG100-115 was reported to inhibit pulmonary eosinophilia andinterleukin-13 as well as mucin accumulation and airwayshyperesponsiveness in a murine model, when administered byaerosolisation. The same authors also reported that the compound wasable to inhibit pulmonary neutrophilia elicited by either LPS orcigarette smoke [Doukas, J. et al., J Pharmacol. Exp. Ther., 2009,328:758-765]

Since it is also activated by oxidative stress, the PI3 kinase δ isoformis likely to be relevant as a target for therapeutic intervention inthose diseases where a high level of oxidative stress is implicated.Downstream mediators of the PI3 kinase signal transduction pathwayinclude Akt (a serine/threonine protein kinase) and the mammalian targetof rapamycin, the enzyme mTOR. Recent work has suggested that activationof PI3 kinase δ, leading to phosphorylation of Akt, is able to induce astate of corticosteroid resistance in otherwise corticosteroid-sensitivecells [To, Y. et al., Am. J. Respir. Crit. Care Med., 2010,182:897-904]. These observations have led to the hypothesis that thissignalling cascade could be one mechanism responsible for thecorticosteroid-insensitivity of inflammation observed in the lungs ofpatients suffering from COPD, as well as those asthmatics who smoke,thereby subjecting their lungs to increased oxidative stress. Indeed,theophylline, a compound used in the treatment of both COPD and asthma,has been suggested to reverse steroid insensitivity through mechanismsinvolving interaction with pathways controlled by PI3 kinase δ [To, Y.et al., Am. J. Respir. Crit. Care Med., 2010, 182:897-904].

At present the mainstay of treatment for both asthma and COPD is inhaledtherapy, using a combination of corticosteroids, muscarinic antagonistsand β₂-agonists, as judged clinically appropriate. One way of addressingthe unmet medical needs in COPD and asthma is to identify newtherapeutic agents, for example suitable for use as inhaled medicines,which have the potential to provide significant benefit when used as amonotherapy or in combination with one or more medicaments from thesethree pharmacological classes. Therefore, there remains a need toidentify and develop isoform selective PI3 kinase inhibitors which havethe potential to provide enhanced therapeutic efficacy in asthma, COPDand other inflammatory diseases.

SUMMARY OF THE INVENTION

According to the invention, there is provided a compound of formula (I):

that is6-(2((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamideor a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof.

The compound of the present disclosure is a dual PI3K delta PI3K gammainhibitor.

The term inhibitor as employed herein is intended to refer to a compoundthat reduces (for example by at least 50%) or eliminates the biologicalactivity of the target protein, for example the PI3K delta isozyme, inan in vitro enzyme assay.

The term delta/gamma inhibitor as employed herein is intended to referto the fact that the compound inhibits, to some degree, both enzymeisoforms although not necessarily to the same extent.

The compound of the present disclosure is active in cell based screeningsystems and thereby demonstrates that it possesses suitable propertiesfor penetrating cells and thereby exert intracellular pharmacologicaleffects.

The compound of the present disclosure has therapeutically relevant anddesirable pharmaceutical properties, for example adequate stability,solubility and potent activity.

In one embodiment there is a provided a pharmaceutically acceptable acidaddition salt of the compound of the invention.

The pharmaceutically acceptable acid addition salts as mentionedhereinabove are meant to comprise the therapeutically active, non-toxic,acid addition salts that the compound of formula (I) is able to form.These pharmaceutically acceptable acid addition salts can convenientlybe obtained by treating the free base form of the compound of formula(I) with such appropriate acid. Appropriate acids comprise, for example,inorganic acids such as hydrochloric acid, hydrobromic acid, andsulfuric, and phosphoric acids and the like; or organic acids such as,for example, acetic, propanoic, hydroxyacetic, lactic, malonic,succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic,para-toluenesulfonic, cyclamic, salicylic, para-aminosalicylic, pamoicacid and the like.

Examples of salts of compound (I) include all pharmaceuticallyacceptable salts, such as, without limitation, acid addition salts ofmineral acids such as HCl and HBr salts and addition salts of organicacids such as a methanesulfonic acid salt. Further examples includesulphuric acid salts and phosphoric acid salts.

In one embodiment there is provided6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamidehydrochloride.

In one embodiment there is provided6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamidehydrobromide.

In one embodiment there is provided6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamidetosylate.

The disclosure also extends to solvates of the compounds herein.Examples of solvates include hydrates.

The compounds of the disclosure include those where the atom specifiedis a naturally occurring or non-naturally occurring isotope. In oneembodiment the isotope is a stable isotope. Thus the compounds of thedisclosure include, for example those containing one or more deuteriumatoms in place of hydrogen atoms and the like.

In one embodiment of the invention wherein the compound of formula (I)is a deuterium labelled compound, the isotopically labelled compound isthe hexadeuterio derivative of formula (IA).

The disclosure also extends to all polymorphic forms of the compoundsherein defined.

The compound of formula (I) may be conveniently prepared by a processcomprising reacting a compound of formula (II):

or a protected derivate thereof wherein LG₁ represents a leaving groupsuch as halo, in particular bromo, with a compound of formula (III):

in the presence of a suitable catalyst and an organic base and in apolar aprotic solvent under an inert atmosphere. Suitable catalystsinclude palladium catalysts such as bis(triphenylphosphine)palladium(II) dichloride, in the presence of copper iodide and a suitable polaraprotic solvent is DMF. A suitable inert atmosphere is nitrogen.

Alternatively the compound of formula (I) may be prepared by a processcomprising of reacting a compound of formula (II) or a protectedderivative thereof with a compound of formula (IV):

wherein R is suitably H to provide a compound of formula (V) or aprotected derivative thereof. The compound of formula (I) is thenobtained from the compound of formula (V) by one or more standardfunctional group transformations. For example where R is H the compoundof formula (I) may be generated from the compound of formula (V) by anamide coupling reaction with an amine, most suitably withbis(2-methoxyethyl)amine.

For synthetic processes in which the compound of formula (II) is aprotected derivative, the compound of formula (I) is revealed by anappropriate deprotection step, as is well known and practiced in theart. For example when the phenol present in the compound of formula (I)is protected with a silyl group, for example with atert-butyldimethylsilyl group the deprotection step can be effected bytreatment with a reagent such as tetrabutylammonium fluoride in thepresence of a polar aprotic solvent such as DMF. The reaction may beperformed at a reduced temperature, such as about 0° C.

Compounds of formula (II) can be prepared by reacting a compound offormula (VI):

or a protected derivative thereof, wherein LG₁ is a leaving group, asdefined hereinabove for compounds of formula (II) and LG₂ is also aleaving group such as halo, for example a halogen atom and suitably achlorine, with a compound of formula (VII):

or a protected derivative thereof, in the presence of a base and in apolar aprotic solvent.

Suitable bases for this transformation include potassium carbonate and asuitable polar aprotic solvent is DMF.

Synthetic processes include those for which is deemed advantageous toprotect the phenolic hydroxyl of the compound of formula (VII) duringthe coupling step and suitable protected derivatives include atertbutyldimethylsilyl ether and a tert-butyl ether.

Alternatively compounds of formula (II) can be prepared by reacting acompound of formula (VIII):

or a protected derivative thereof, wherein LG₁, is as defined above forcompounds of formula (II) and LG₃ represents a leaving group such ashalo, in particular iodo, with a compound of formula (IX):

or a protected derivate thereof, in the presence of a suitable noblemetal catalyst, an inorganic base and a polar protic solvent, under aninert atmosphere; followed, where appropriate, by deprotection.

A suitable catalyst is tetrakis(triphenylphosphine)palladium(0).

A suitable inorganic base is sodium carbonate and a suitable polarprotic solvent is ethanol.

The reaction may be performed at an elevated temperature, for example at85° C. for an extended period such as, for example, 3 days beforecooling to RT.

Protecting groups may be advantageous to mask chemically sensitivegroups during one or more of the reaction sequences described above, toensure that one or more of the processes are efficient. Thus if desiredor necessary, intermediate compounds may be protected by the use ofconventional protecting groups. Protecting groups and means for theirremoval are described in “Protective Groups in Organic Synthesis”, byTheodora W. Greene and Peter G. M. Wuts, published by John Wiley & SonsInc; 4^(th) Rev Ed., 2006, ISBN-10: 0471697540.

Novel intermediates are claimed as an aspect of the invention.

Advantageously, compounds of the present invention do not exhibitatropisomerism.

In one aspect the compound is useful in treatment, for example COPDand/or asthma.

The PI3K compounds developed to date have typically been intended fororal administration. Typically this strategy involves the optimisationof a compound's pharmacokinetic profile in order to achieve an adequateduration of action. In this way a sufficiently high drug concentrationis established and maintained between doses to provide continuousclinical benefit. An inevitable and frequently undesired consequence ofthis approach is that non-targeted body tissues, especially the liverand the gut, are likely to be exposed to pharmacologically activeconcentrations of the drug.

An alternative strategy is to design treatment regimens in which thedrug is dosed directly to the inflamed organ (for example topicaltherapy). Although this approach is not suitable for treating allchronic inflammatory conditions, it has been extensively exploited intreating lung diseases (asthma, COPD), skin lesions (atopic dermatitisand psoriasis), nasal diseases (allergic rhinitis) and gastrointestinaldisorders (ulcerative colitis).

In topical therapy, the desired efficacy can sometimes be achieved byensuring that the drug has a sustained duration of action and isretained predominantly in the target organ, thereby minimising the risksof systemic toxicity. Alternatively an appropriate formulation can beused which generates a “reservoir” of the active drug which is thenavailable to sustain the desired effects. The first approach isexemplified in the use of the anticholinergic drug tiotropium bromide(Spiriva HandiHaler®), which is administered topically to the lung as atreatment for COPD. This compound has an exceptionally high affinity forits target receptor resulting in a very slow off rate (dissociationrate) and a consequent sustained duration of action.

There is provided according to one aspect of the present disclosure useof the compound of formula (I) or a suitable formulation derivedtherefrom, as a PI3 kinase inhibitor, for example administered topicallyto the lung.

In one aspect of the disclosure the compound herein is particularlysuitable for topical delivery, such as topical delivery to the lungs, inparticular for the treatment of COPD.

Thus is one aspect there is provided use of a compound of the inventionfor the treatment of COPD and/or asthma, in particular COPD or severeasthma, by inhalation i.e. by topical administration to the lung.Advantageously, administration to the lung allows the beneficial effectsof the compounds to be realised whilst minimising the side-effects, forpatients.

In one embodiment the compound is suitable for sensitizing patients totreatment with a corticosteroid.

The compound herein disclosed may also be useful for the treatment ofrheumatoid arthritis.

Further, the present invention provides a pharmaceutical compositioncomprising a compound according to the disclosure optionally incombination with one or more pharmaceutically acceptable diluents orcarriers.

Diluents and carriers may include those suitable for parenteral, oral,topical, mucosal and rectal administration, and may be differentdepending on the route of administration.

In one embodiment compositions may be prepared e.g. for parenteral,subcutaneous, intramuscular, intravenous, intra-articular orperi-articular administration, particularly in the form of liquidsolutions or suspensions; for oral administration, particularly in theform of tablets or capsules; for topical e.g. pulmonary or intranasaladministration, particularly in the form of powders, nasal drops oraerosols and transdermal administration; for mucosal administration e.g.to buccal, sublingual or vaginal mucosa, and for rectal administratione.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985).

Formulations for parenteral administration may contain as excipientssterile water or saline, alkylene glycols such as propylene glycol,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, hydrogenated naphthalenes and the like.

Formulations for nasal administration may be solid and may containexcipients, for example, lactose or dextran, or may be aqueous or oilysolutions for use in the form of nasal drops or metered spray. Forbuccal administration typical excipients include sugars, calciumstearate, magnesium stearate, pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or morephysiologically compatible carriers and/or excipients and may be insolid or liquid form. Tablets and capsules may be prepared with bindingagents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, orpoly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch,calcium phosphate, sorbitol, or glycine; lubricants, such as magnesiumstearate, talc, polyethylene glycol, or silica; and surfactants, such assodium lauryl sulfate. Liquid compositions may contain conventionaladditives such as suspending agents, for example sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or ediblefats; emulsifying agents such as lecithin, or acacia; vegetable oilssuch as almond oil, coconut oil, cod liver oil, or peanut oil;preservatives such as butylated hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT). Liquid compositions may be encapsulated in, forexample, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsulesand soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40% to 60%concentration of gelatin, about a 20% to 30% concentration ofplasticizer (such as glycerin, sorbitol or propylene glycol) and about a30% to 40% concentration of water. Other materials such aspreservatives, dyes, opacifiers and flavours also may be present. Theliquid fill material comprises a solid drug that has been dissolved,solubilized or dispersed (with suspending agents such as beeswax,hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug invehicles or combinations of vehicles such as mineral oil, vegetableoils, triglycerides, glycols, polyols and surface-active agents.

Suitably the compound of formula (I) is administered topically to thelung. Hence in one embodiment there is provided a pharmaceuticalcomposition comprising a compound of the disclosure optionally incombination with one or more topically acceptable diluents or carriers.Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane (propellant 11),dichlorotetrafluoromethane (propellant 114), and dichlorodifluoromethane(propellant 12). Suitable HFC propellants include tetrafluoroethane(HFC-134a) and heptafluoropropane (HFC-227). The propellant typicallycomprises 40% to 99.5% e.g. 40% to 90% by weight of the total inhalationcomposition. The formulation may comprise excipients includingco-solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitantrioleate and the like). Aerosol formulations are packaged in canistersand a suitable dose is delivered by means of a metering valve (e.g. assupplied by Bespak, Valois or 3M).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.This may be administered by means of a nebuliser. Topical administrationto the lung may also be achieved by use of a dry-powder formulation. Adry powder formulation will contain the compound of the disclosure infinely divided form, typically with a mass mean diameter (MMAD) of 1-10μm. The formulation will typically contain a topically acceptablediluent such as lactose, usually of large particle size e.g. a mass meandiameter (MMAD) of 100 μm or more. Example dry powder delivery systemsinclude SPINHALER, DISKHALER, TURBOHALER, DISKUS, SKYEHALER, ACCUHALERand CLICKHALER.

In one embodiment a compound of the present invention is provided as amicronized dry powder formulation, for example comprising lactose of asuitable grade, filled into a device such as DISKUS.

The compound according to the disclosure is intended to have therapeuticactivity. In a further aspect, the present invention provides a compoundof the disclosure for use as a medicament.

The compound according to the disclosure may also be useful in thetreatment of respiratory disorders including COPD (including chronicbronchitis and emphysema), asthma, paediatric asthma, cystic fibrosis,sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis,sinusitis, especially asthma, chronic bronchitis and COPD.

The compound of the disclosure may also re-sensitise the patient'scondition to treatment with a corticosteroid, when previously thepatient's condition had become refractory to the same.

In one embodiment of the invention a dose of the present compound isemployed that is equal to that suitable for use as a monotherapy butadministered in combination with a corticosteriod.

In one embodiment a dose of the compound of formula (I) that would besubtherapeutic as a single agent is employed, in combination with acorticosteriod, thereby restoring patient responsiveness to the latter,in instances where the patient had previously become refractory to thesame.

Additionally, the compound of the disclosure may exhibit anti-viralactivity and prove useful in the treatment of viral exacerbations ofinflammatory conditions such as asthma and/or COPD.

The compound of the present disclosure may also be useful in theprophylaxis, treatment or amelioration of flu virus, rhinovirus and/orrespiratory syncytical virus.

The compound of formula (I), according to the disclosure is alsoexpected to be useful in the treatment of certain conditions which maybe treated by topical or local therapy including allergicconjunctivitis, conjunctivitis, allergic dermatitis, contact dermatitis,psoriasis, ulcerative colitis, inflamed joints secondary to rheumatoidarthritis or osteoarthritis.

In one embodiment the compound of formula (I) is considered useful inthe, treatment of Hepatitis C and/or HIV, when administered by anappropriate route. Appropriate routes of administration may includeoral, intravenous injection or infusion.

In one embodiment a compound of formula (I) for the treatment ofHepatitis C is delivered to the blood pre-entry to the liver.

The compound of the disclosure is also expected to be useful in thetreatment of certain other conditions including rheumatoid arthritis,pancreatitis, cachexia, inhibition of the growth and metastasis oftumours including non-small cell lung carcinoma, breast carcinoma,gastric carcinoma, colorectal carcinomas and malignant melanoma.

In one embodiment the presently disclosed compound and pharmaceuticalformulations comprising the same are useful in the treatment orprevention of cancer, in particular lung cancer, especially by topicaladministration to the lung.

Thus, in a further aspect, the present invention provides a compound asdescribed herein for use in the treatment of one or more of the abovementioned conditions.

In a further aspect, the present invention provides use of a compound asdescribed herein for the manufacture of a medicament for the treatmentof one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method oftreatment of the above mentioned conditions which comprisesadministering to a subject an effective amount of a compound of thedisclosure or a pharmaceutical composition thereof.

Compounds described herein may also be used in the manufacture of amedicament for the treatment of one or more of the above-identifieddiseases.

The word “treatment” is intended to embrace prophylaxis as well astherapeutic treatment.

A compound of the disclosure may also be administered in combinationwith one or more other active ingredients e.g. active ingredientssuitable for treating the above mentioned conditions. For examplepossible combinations for treatment of respiratory disorders includecombinations with steroids (e.g. budesonide, beclomethasonedipropionate, fluticasone propionate, mometasone furoate, fluticasonefuroate), beta agonists (e.g. terbutaline, salbutamol, salmeterol,formoterol, indacaterol) and/or xanthines (e.g. theophylline),musacarinic antagonists, (e.g. ipratropium) and/or a p38 MAP kinaseinhibitor.

In one embodiment a compound of the disclosure is administered incombination with an antiviral agent, for example acyclovir, tamiflu,relenza or interferon.

In one embodiment the combination of active ingredients areco-formulated.

In one embodiment a compound of the present disclosure is co-formulatedwith a corticosteriod as a formulation for inhalation, for example foruse in maintenance therapy of COPD or lung cancer including preventionof the latter.

In one embodiment the combination of active ingredients is simplyco-administered.

In one embodiment the compound of the disclosure is administered byinhalation and a corticosteriod is administered orally or by inhalationeither in combination or separately.

EXPERIMENTAL SECTION

Abbreviations used herein are defined below (Table 1). Any abbreviationsnot defined are intended to convey their generally accepted meaning.

TABLE 1 Abbreviations aq aqueous Ac acetyl ATP adenosine-5′-triphosphateBALF bronchoalveolae lavage fluid br broad BSA bovine serum albumin COPDchronic obstructive pulmonary disease d doublet DCM dichloromethane DMAP4-dimethylaminopyridine DMSO dimethyl sulfoxide EDC•HCl1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (ES⁺)electrospray ionization, positive mode Et ethyl EtOAc ethyl acetate EtOHethanol FACS fluorescence-activated cell sorting FCS foetal calf serumFP fluticasone propionate g gram(s) HPLC-MS high performance liquidchromatography mass spectrometry hr hour(s) HRP horseradish peroxidaseHRV human rhinovirus i-n intra-nasal i-t intra-tracheal IL-8 interleukin8 μL microliter(s) LPS lipopolysaccharide μM micromolar M molar (M + H)⁺protonated molecular ion MCP-1 monocyte chemoattractant protein Memethyl MeOH methanol mg milligram(s) MHz megahertz min minute(s) mLmillilitre(s) mM millimolar mmol millimole(s) MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide m/zmass-to-charge ratio ng nanogram nM nanomolar nm nanometre NMR nuclearmagnetic resonance (spectroscopy) OVA ovalbumin PBS phosphate bufferedsaline Ph phenyl PIP2 phosphatidylinositol 4,5-biphosphate PIP3phosphatidylinositol 3,4,5-triphosphate PMA phorbol myristate acetate poby oral administration PPh₃ triphenylphosphine q quartet quin quintetR^(t) retention time RT room temperature RP HPLC reverse phase highperformance liquid chromatography RSV respiratory syncytial virus ssinglet SDS sodium dodecyl sulfate SEM standard error of the mean ttriplet TMB 3,3′,5,5′-tetramethylbenzidine TNFα tumour necrosis factoralpha TR-FRET time-resolved fluorescence resonance energy transfer volvolume

General Procedures

All starting materials and solvents were obtained either from commercialsources or prepared according to the literature citation. Unlessotherwise stated all reactions were stirred. Organic solutions wereroutinely dried over anhydrous magnesium sulfate.

HPLC-MS was performed on Agilent HP1200 systems using Agilent Extend C18columns, (1.8 μm, 4.6×30 mm) at 40° C. and a flow rate of 2.5-4.5 mLmin⁻¹, eluting with a H₂O—MeCN gradient containing 0.1% v/v formic acidover 4 min. Gradient information: 0-3.00 min, ramped from 95% H₂O-5%MeCN to 5% H₂O-95% MeCN; 3.00-3.01 min, held at 5% H₂O-95% MeCN, flowrate increased to 4.5 mL min⁻¹; 3.01-3.50 min, held at 5% H₂O-95% MeCN;3.50-3.60 min, returned to 95% H₂O-5% MeCN; flow rate reduced to 3.50 mLmin⁻¹; 3.60-3.90 min, held at 95% H₂O-5% MeCN; 3.90-4.00 min, held at95% H₂O-5% MeCN, flow rate reduced to 2.5 mL min⁻¹. UV detection wasperformed at 254 nm using an Agilent G1314B variable wavelengthdetector.

Mass spectra were obtained using electrospray ionization (ES) over therange m/z 60 to 2000 at a sampling rate of 1.6 sec/cycle using anAgilent G1956B, over m/z 150 to 850 at a sampling rate of 2 Hz using aWaters ZMD or over m/z 100 to 1000 at a sampling rate of 2 Hz using aShimadzu 2010 LC-MS system. ¹H NMR spectra were acquired on a BrukerAvance III spectrometer at 400 MHz using residual undeuterated solventas reference.

5-Bromo-3-(2-chlorobenzyl)-2-(chloromethyl)quinazolin-4(3H)-one

To a stirred solution of 2-amino-6-bromo-benzoic acid (3.06 g, 14.2mmol) in toluene (75 mL) cooled to 0° C. in an ice-bath was addedpyridine (0.60 mL, 7.10 mmol) followed by a solution of chloroacetylchloride (2.26 mL, 28.4 mmol) in toluene (75 mL) drop-wise over 1 hr.The reaction mixture was allowed to warm to RT, and was heated at 115°C. for 3 hr and then allowed to cool to RT. The solvent volume wasreduced by half by evaporation in vacuo. Upon standing overnight, theproduct precipitated and was collected by filtration to afford2-bromo-6-(2-chloroacetamido)benzoic acid (1a, X═Cl) (1.44 g) as a whitesolid: m/z 290/292 (M+H)⁺ (ES⁺). The filtrate was concentrated in vacuoand the residue triturated with ethanol/heptane to afford2-bromo-6-(2-hydroxyacetamido) benzoic acid (1b X═OH) (1.02 g, combinedyield, 59%): m/z 274/276 (M+H)⁺ (ES⁺). Both 1a and 1b can be usedwithout further purification in the next step.

To a stirred mixture of compound (1a) (7.50 g, 27.4 mmol),2-chlorobenzylamine (5.00 mL, 41.05 mmol) and triethylamine (5.70 mL,41.1 mmol) in toluene (250 mL) was added a solution of phosphorustrichloride (2.60 mL, 30.1 mmol) in toluene (250 mL) dropwise over 1 hr.The reaction mixture was heated to 110° C. for 24 hr, whereupon the hotsolution was decanted and concentrated in vacuo. The residue wastriturated with propan-2-ol (50 mL) to afford the title compound (2)(6.41 g, 59%) as a yellow solid: R^(t) 2.67 min; m/z 397/399 (M+H)⁺(ES⁺).

24(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-bromo-3-(2-chlorobenzyl)quinazolin-4(3H)-one

To a stirred mixture of5-bromo-3-(2-chlorobenzyl)-2-(chloromethyl)quinazolin-4(3H)-one, (2),(13.6 g, 30.7 mmol) and 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3)(8.09 g, 30.7 mmol) in DMF (300 mL was added potassium carbonate (6.36g, 46.0 mmol) and the reaction maintained at RT in the dark for 24 hr.The mixture was poured onto water (4.0 L) and the resulting suspensionwas stirred at RT for 1 hr. The precipitate was isolated by filtrationand dried in vacuo to afford the title compound, (4), as a colourlesssolid (18.0 g, 94%); R^(t) 2.17 min; m/z 622/624 [M+H]⁺ (ES⁺).

3-(3-(tert-Butyldimethylsilyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a stirred suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine(3) (8.22 g, 31.5 mmol), 3-phenol boronic acid (13.0 g, 94.5 mmol) andpotassium phosphate (10.0 g, 47.3 mmol) in degassed DMF/water (3:2, 140mL) was added [dppf] palladium (II) dichloride (13.0 g, 15.7 mmol). Thereaction mixture was flushed with nitrogen, heated at 120° C. for 2 hrand then allowed to cool to RT. The reaction mixture was diluted withEtOAc (500 mL) and hydrochloric acid (2 M, 500 mL) and the resultingsuspension was filtered. The filtrate was extracted with hydrochloricacid (2 M, 2×500 mL). The combined aq extracts were basified with asaturated aq solution of sodium carbonate to pH 10. The precipitateformed was filtered and the filtrate was extracted with EtOAc (3×1 L).The combined organic extracts were dried, filtered and the solventremoved in vacuo to afford a grey solid. All solid materials generatedduring the workup procedure were combined and triturated with DCM toafford 3-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (5) (6.04 g,84%) as a grey solid: m/z 228 (M+H)⁺ (ES⁺).

To a stirred solution of the phenol (5) (4.69 g, 20.66 mmol) andimidazole (2.10 g, 30.99 mmol) in dry DMF (100 mL) was added TBDMSCI(4.70 g, 30.99 mmol). After 16 hr, further aliquots of imidazole (2.10g, 30.99 mmol) and TBDMSCI (4.70 g, 30.99 mmol) were added and themixture was stirred for 48 hr. The reaction mixture was diluted withwater (120 mL) and extracted with DCM (2×200 mL). The combined organicextracts were washed with water (2×200 mL), dried, filtered and thevolume reduced to approximately 100 mL by evaporation in vacuo. Theresulting slurry was filtered and the solid washed with heptane (50 mL)to afford the title compound (6) (6.05 g, 85%) as an off-white solid:m/z 343 (M+H)⁺ (ES⁺).

Intermediate A:2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-bromo-3-(2-chlorobenzyl)quinazolin-4(3H)-one

To a stirred mixture of5-bromo-3-(2-chlorobenzyl)-2-(chloromethyl)quinazolin-4(3H)-one (2) (100mg, 0.25 mmol) and potassium carbonate (42 mg, 0.30 mmol) in DMF (2.5mL) was added a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine(3) (94 mg, 0.28 mmol) in DMF (2.5mL) and the reaction mixture wasstirred at RT for 18 hr. Potassium carbonate (3×35 mg, 0.75 mmol) wasadded in three portions over 30 hr. after which the solvent was removedin vacuo and the crude material was purified by flash columnchromatography, eluting with 4.5% methanol in DCM, to afford the titlecompound, Intermediate A, (94 mg, 64%) as a off-white solid: R^(t) 2.01min; m/z 588/590 (M+H)⁺, (ES⁺).

Intermediate B: N,N-bis(2-Methoxyethyl)hex-5-ynamide

To a solution of hex-5-ynoic acid (7.11 g, 63.4 mmol), EDC.HCl (14.0 g,72.9 mmol) and DMAP (387 mg, 3.17 mmol) in DCM (600 mL) at 0° C. wasadded bis(2-methoxyethyl)amine (9.3 mL, 63 mmol). The resulting mixturewas warmed to RT for 20 hr and was then washed with hydrochloric acid (1M, 2×500 mL) and water (500 mL). The organic layer was dried andevaporated in vacuo to afford the title compound, Intermediate B, as ayellow oil (16 g, 97%): ¹H NMR (400 MHz, CDCl₃) δ: 1.88 (3H, m), 2.26(2H, m), 2.49 (2H, m), 3.32 (6H, s), 3.51 (4H, m), 3.55 (4H, m)

6-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide:Compound of Formula (I)

A mixture of Intermediate B (9.11 g, 34.9 mmol), bis-triphenylphosphinepalladium(II) dichloride (0.98 g, 1.4 mmol), Intermediate A (8.3 g, 14mmol), and copper(I) iodide (0.27 g, 1.4 mmol), in diethylamine (400 mL,3.8 mol) was degassed with nitrogen and was then stirred at 60° C. for 4hr then cooled to RT for a further 72 hr. The mixture was evaporated invacuo and the residue was partitioned between aq ammonium acetate (500mL) and EtOAc (500 mL). The organic layer was separated and was washedwith brine (2×500 mL) and then dried and evaporated in vacuo. Theresidue was purified by flash column chromatography, (SiO₂, 120 g, MeOHin DCM, 0-5%, gradient elution) to afford the title compound of formula(I), as an off white solid (6.9 g, 66%): R^(t) 1.92 min; m/z 735/737(M+H)⁺ (ES⁺) (Method D); ¹H NMR (400 MHz, DMSO-d₆) δ: 1.70 (2H, quin),2.46 (2H, t), 2.55 (2H, t), 3.12 (3H, s), 3.19 (3H, s), 3.26 (2H,overlapping m), 3.31 (4H, m, partially obscured by HOD peak), 3.35 (2H,q), 5.30 (2H, s), 5.76 (2H, s), 6.18 (1H, dd), 6.80 (1H, dt), 6.85 (1Hddd), 6.92-6.94 (2H, overlapping m), 7.05 (1H, td), 7.13 (1H, dd), 7.31(1H, t), 7.61 (1H, dd), 7.68 (1H, dd), 7.81 (1H, dd), 8.18 (1H, br s),9.65 (1H, br s).

The additional complexity and consequences for drug developmentresulting from atropisomerism are analogous to those arising from othersources of molecular isomerism such as the presence of a stereogeniccentre. This property renders such molecules both chiral, and unlessresolved, a racemic mixture; the components of which could possessdifferent pharmacological and toxicological profiles. This feature islikely to significantly increase downstream development costs for suchmolecules, and the absence of atropisomerism in the compound of formula(I) disclosed herein is therefore a highly desirable and advantageousproperty.

Biological Testing: Experimental Methods

Enzyme Inhibition Assay

PI3 kinases catalyse the phosphorylation of phosphatidylinositol4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3)in the presence of ATP and Mg²⁺ ions. The PIP3 product can be detectedby displacement of biotin-PIP3 from energy transfer complexes consistingof europium labelled anti-GST monoclonal antibody, a GST-taggedPleckstrin homology (PH) domain, biotinylated PIP3 andstreptavidin-allophycocyanin (APC) by the time-resolved fluorescenceresonance energy transfer (TR-FRET) (HTRF®PI3K enzyme assay, Millipore).Excitation (330 nm) of europium in the complex results in an energytransfer to the APC and a fluorescent emission at 665 nm althougheuropium itself emits at its characteristic 620 nm. The PIP3 productformed by PI3K activity displaces biotin-PIP3 from the complex andresults in a loss of energy transfer (decreasing signal).

The compound to be tested was added, at the desired finalconcentrations, to a mixture of PIP2 substrate and recombinant PI3kinase α, δ or γ enzymes (Millipore), and the mixture incubated for 2 hrat RT. Following this incubation period, ATP (20 μM) was added to theenzyme/compound/PIP2 substrate mixture and the resulting mixture wasincubated for 30 min at RT. A stopping solution containing biotinylatedPIP3 and the detection mix containing the GST tagged GRP1 pleckstrinhomology (PH) domain and fluorophores were then added and the mixturewas incubated at RT for 15-18 hr, prior to detection in a fluorescencemicroplate reader (Varioskan® Flash, ThermoFisher Scientific).

The results were calculated according to the formula: APC signal(emission at 665 nm)/europium signal:(emission at 620 nm)×10⁴. Thepercentage inhibition of each reaction was calculated relative to DMSOtreated control, and the 50% inhibitory concentration (IC₅₀ value) thencalculated from the concentration-response curve.

PI3Kδ Cell Based Assay

As a means of assessing PI3K δ activation in response to stimuli, thephosphorylation status of the protein, Akt, a downstream product ofPI3Kδ, signaling was determined.

Human monocytic cells (U937 cells), were differentiated tomacrophage-type cells by incubation with PMA (100 ng/mL) for 48 to 72hr. Cells were then pre-incubated with either the test compound orvehicle for 2 hr and were then stimulated briefly by exposure to H₂O₂(10 mM; 5-7 min) and the reaction stopped by replacing the media with 4%formaldehyde solution. Endogenous peroxide activity and formaldehydewere inactivated by incubating with quenching buffer (0.1% sodium azide,1% H₂O₂ in PBS with 0.1% Triton X-100) for 20 min. The cells were washedwith buffer (PBS containing 0.1% Triton X-100) and were incubated withblocking solution (1% BSA in PBS) for 1 hr and were then re-washed withbuffer and incubated overnight with either anti-pAkt antibody oranti-pan-Akt antibody (both from Cell Signaling Technology). Afterwashing with buffer (PBS containing 0.1% Triton X-100), cells wereincubated with an HRP-conjugated secondary antibody (Dako) and theresultant signal was determined colorimetrically (OD: 450 nm with areference wavelength of 655 nm) using TMB substrate (substrate reagentpack supplied by R&D Systems, Inc.).

This reaction was stopped by addition of 100 μL of 1N H₂SO₄ solution.Cells were then washed with buffer (PBS containing 0.1% Triton X-100)and 100 μL of 5% crystal violet solution was applied for 30 min. Afterwashing with buffer (PBS containing 0.1% Triton X-100) 100 μL of 1% SDSwas added to each well and the plates were shaken lightly for 1 hr priorto measuring the absorbance at 595 nm (Varioskan® Flash, Thermo-FisherScientific). The measured OD₄₅₀₋₆₅₅ readings were corrected for cellnumber by dividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅ readings. The ratio ofpAkt signal to total Akt signal was used to quantitate the extent ofPI3K δ activation. The percentage inhibition for each well wascalculated relative to a 10 μg/mL standard control (LY294002) set to100% inhibition versus H₂O₂-only controls as 0% inhibition. The IC₅₀values were calculated from the concentration-response curves generatedby the serial dilutions of the test compounds.

PI3K γ Cell Based Assay

As a means of assessing the activation of PI3K γ in response to stimuli,the phosphorylation status of the protein, Akt, a downstream product ofPI3K γ, signalling was determined following stimulation with MCP-1.

Human monocytic cells (THP1 cells) were pre-incubated in 1% FCSRPMI-1640 media for 1 hr. Cells were then treated with either the testcompound or vehicle for 1 hr and stimulated briefly by exposure to MCP-1(50 nM, 5-7 min; R&D systems, MN, USA). The reaction was stopped byreplacing the media with 4% formaldehyde solution followed bypermeabilisation using IntraPrep™ (Beckman Coulter, France) followingthe manufacturer's instructions. The cells were washed with washingbuffer (PBS containing 0.1% BSA) and then incubated withanti-phospho-Akt (#9271; Cell Signaling, Danvers, Mass., USA) for 15 minat RT. After washing with washing buffer, cells were incubated with aPacific blue-conjugated goat anti-rabbit antibody (Life TechnologiesCorp., Carlsbad, Calif., USA) and the fluorescence level was determinedusing an ATTUNE flow cytometer (Life Technologies Corp.). From thehistogram, the minimum percentage of positive cells was calculated ineach sample compared with baseline control and used to quantitate theextent of PI3K γ activation. The percentage inhibition for each well wascalculated relative to a 10 μg/mL standard control (LY294002) set to100% inhibition versus MCP1-only controls as 0% inhibition. The IC₅₀value was calculated from the concentration-response curves generated bythe serial dilutions of the test compounds.

Rhinovirus-Induced IL-8 Release

Human rhinovirus RV16 was obtained from the American Type CultureCollection (Manassas, Va.). Viral stocks were generated by infectingHela cells with HRV until 80% of the cells were cytopathic. BEAS2B cellswere infected with HRV at an MOI of 5 and incubated for 2 hr at 33° C.with gentle shaking to promote absorption. The cells were then washedwith PBS, fresh media was added and the cells were incubated for afurther 72 hr. The supernatant was collected for assay of IL-8concentrations using a Duoset ELISA development kit (R&D systems,Minneapolis, Minn.).

In Vitro RSV Virus Load in Primary Bronchial Epithelial Cells

NHBEC (normal human bronchial epithelial cells) grown in 96 well plateswere infected with RSV A2 (Strain A2, HPA, Salisbury, UK; at an MOI of0.001) in the LHC8 Media:RPMI-1640 (50:50) containing 15 mM magnesiumchloride and incubated for 1 hr at 37° C. for adsorption. The cells werethen washed with PBS, fresh media was added and the cells were incubatedfor 4 days. Where appropriate, cells were pre-incubated with the testcompound or DMSO for 2 hr, and then added again after washout of virus.

The cells were fixed with 4% formaldehyde in PBS solution for 20 min,washed with washing buffer (PBS including 0.5% BSA and 0.05% Tween-20)and incubated with blocking solution (5% condensed milk in PBS) for 1hr. Cells were then washed with washing buffer and incubated for 1 hr atRT with anti-RSV (2F7) F-fusion protein antibody (mouse monoclonal; lot798760, Cat. No. ab43812, Abcam). After washing, cells were incubatedwith an HRP-conjugated secondary antibody (lot 00053170, Cat. No. P0447,Dako) and then TMB substrate (substrate reagent pack lot 269472, Cat.No. DY999, R&D Systems, Inc.) was added. This reaction was stopped byaddition of 2N H₂SO₄ (50 μL) and the resultant signal was determinedcolorimetrically (OD: 450 nm with a reference wavelength of 655 nm) in amicroplate reader (Varioskan® Flash, ThermoFisher Scientific). Cellswere then washed and a 2.5% crystal violet solution (lot 8656, Cat. No.PL7000, Pro-Lab Diagnostics) was applied for 30 min. After washing withwashing buffer, 100 μL of 1% SDS was added to each well, and plates wereshaken lightly on the shaker for 1 hr prior to reading the absorbance at595 nm. The measured OD₄₅₀₋₆₅₅ readings were corrected to the cellnumber by dividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅ readings. The percentageinhibition for each well was calculated and the IC₅₀ value wascalculated from the concentration-response curve generated by the serialdilutions of compound.

MTT Assay

PMA-differentiated U937 cells were pre-incubated with test compound for4 hr in 5% FCS or 10% FCS for 24 hr. The supernatant was replaced with200 μL of new media and 10 μL of MTT stock solution (5 mg/mL) added toeach well. After 1 hr incubation, the media were removed, 200 μL of DMSOadded to each well and the plates were shaken lightly for 1 hr prior toreading the absorbance at 550 nm. The percentage loss of cell viabilitywas calculated for each well relative to vehicle (0.5% DMSO)-treatment.

In Vivo Screening: Pharmacodynamics and Anti-Inflammatory Activity

Ovalbumin-Induced Nasal Eosinophil and Neutrophil Accumulation in Mice

BALB/c mice (6-8 weeks old) were immunized with OVA (40 μg/kg i.p.) onday 1 and 5. In order to elicit local inflammatory responses in thenose, mice were repeatedly challenged intra-nasally (10 μL per nostril)on days 12-19 with OVA (3% OVA in saline). On day 19 non-fasted micewere dosed intra-nasally (10 μL/nostril) with either vehicle or testcompound at T=−2 hr relative to the start of the final OVA challenge. AtT=0, each animal received a final intranasal OVA (3%) challenge. After afurther 8 hr, each animal was anaesthetized and nasal lavage was carriedout by instilling 1 mL of PBS into the posterior nares via a rostrallyimplanted tracheal cannula extending to a position that wasapproximately 1 mm before the posterior nares. This procedure wasrepeated to give a yield of approximately 2 mL of lavage fluid. Totalcell numbers in the nasal lavage fluid samples were measured using ahaemocytometer. Cytospin smears of the nasal lavage fluid samples wereprepared by centrifugation at 1200 rpm for 2 min at RT and stained usinga DiffQuik stain system (Dade Behring) for differential cell counts.Cells were counted using oil immersion microscopy. Data is expressed asdifferential number of cells per mL of nasal lavage fluid, mean±S.E.M.

Poly-I:C-Induced Cell Accumulation in Mice

Specific pathogen-free A/J mice (males, 5 weeks old) were administeredwith poly (I:C)-LMW (poly-IC; 1 mg/mL, 40 μL, in; InvivoGen, San Diego,Calif., USA) intranasally twice daily for 3 days under anaesthasia with3% isoflurane. Test substances were given intra-nasally (35 μL ofsolution in 50% DMSO/PBS) 2 hr before each poly-I:C treatment. Twentyfour hr after the last poly-I:C challenge, animals were anesthetized,the trachea cannulated and BALF was collected. The concentrations ofalveolar macrophages and neutrophils in BALF were determined by FACSanalysis (EPICS® ALTRA II, Beckman Coulter, Inc., Fullerton, Calif.,USA) using anti-mouse MOMA2 antibody (macrophage) or anti-mouse 7/4antibody (neutrophil).

Cigarette Smoke Model

A/J mice (males, 5 weeks old) were exposed to cigarette smoke (4%cigarette smoke, diluted with compressed air) for 30 min/day for 11 daysusing a Tobacco Smoke Inhalation Experiment System for small animals(Model SIS-CS; Sibata Scientific Technology, Tokyo, Japan). Testsubstances were given intra-nasally (35 μL of solution in 50% DMSO/PBS)and therapeutically twice daily for 3 days after the final cigarettesmoke exposure. Twelve hr after the last dosing, animals wereanesthetized, the trachea cannulated and bronchoalveolar lavage fluid(BALF) was collected. The numbers of alveolar macrophages andneutrophils were determined by FACS analysis (EPICS® ALTRA II, BeckmanCoulter, Inc., Fullerton, Calif., USA) using anti-mouse MOMA2 antibody(macrophage) or anti-mouse 7/4 antibody (neutrophil).

Summary of In Vitro and In Vivo Screening Results

The in vitro profile of the compound of formula (I) disclosed herein, asdetermined using the methods described above is presented below (Tables2 and 3). The compound of the present invention demonstrates potentinhibition of both PI3 kinase δ and γ isoforms, and shows only modestinhibitory activity versus PI3 kinase α in enzyme assays. These effectstranslate into potent inhibiton of Akt phosphorylation induced by thestimulation of cells with either hydrogen peroxide or MCP-1, as well asan inhibitory activity versus HRV-induced IL-8 release and RSV-inducedF-protein expression in epithelial cells. No effects on cell viability,resulting from incubation with the compound of formula (I), weredetected.

TABLE 2 Effects of the compound of formula (I) on PI3K isoforms; oncellular, hydrogen peroxide or MCP-1 induced phosphylation of Akt and oncell viability PI3 Kinase Cellular Activity Inhibition IC₅₀ IC₅₀ valuesfor inhibition Cell Viability value at stated of induced Aktphosphorylation MTT Assay in d- isozyme (nM) H₂O₂ stimulus in MCP-1stimulus U937 cells^(a) δ γ α d-U937 cells in THP1 cells at 4 hr at 24hr 12 25 193 1.1 46^(b) −ve −ve ^(a)−ve indicates a value of <30%inhibition; ^(b)calculated IC₅₀ value by linear regression.

TABLE 3 Effects of the compound of formula (I) on HRV-induced IL-8release and on RSV-induced F-protein expression HRV16-induced IL-8release in RSV F protein expression in bronchial BEAS2B cells (%inhibition) epithelial cells: IC₅₀ value (nM) 78% (at 0.1 μg/mL) 389

Treatment of mice intra-nasally with the compound disclosed herein wasfound to produce a dose-dependent inhibition of both eosinophil andneutrophil accumulation in nasal lavage following allergen challenge(Table 4).

TABLE 4 The effects of treatment with the compound of formula (I) onOVA-induced airway eosinophilia and neutrophilia in mice. Cell numbersin nasal lavage fluid Compound (I) (×10⁵/mL) and (% inhibition) (mg/mL)Eosinophils Neutrophils Vehicle 1.51 ± 0.22 0.34 ± 0.06 0.05 1.13 ± 0.16(25) 0.27 ± 0.04 (21) 0.2 0.57 ± 0.14 (62) 0.15 ± 0.04 (56) N = 8 pergroup

The effect of treatment with the compound of the present invention onmacrophage and neutrophil accumulation in BALF following exposure ofmice to poly-I:C was also investigated. Treatment with compound offormula (I) was found to produce a dose-dependent inhibition ofpoly-I:C-induced macrophage and neutrophil accumulation into BALF (Table5).

TABLE 5 The effects of treatment with the compound of formula (I) onpoly-I:C-induced cell accumulation in mice airways. Treatment and doseof Cell numbers in BAL ×10⁴/mL Compound of formula (I) (% inhibition)(mg/mL) Macrophages Neutrophils Vehicle  5.0 ± 0.90  3.0 ± 0.55Vehicle + Poly I:C 16.7 ± 1.6 11.8 ± 0.3 Poly I:C + (I) (0.002) 13.8 ±0.48 (25) 10.8 ± 0.59 (11) Poly I:C + (I) (0.02) 12.2 ± 0.29 (38)  9.4 ±0.26 (27) Poly I:C + (I) (0.2)  9.4 ± 0.75 (61)  7.9 ± 0.74 (44) PolyI:C + (I) (2)  6.4 ± 1.2 (88)  5.6 ± 0.66 (70) The data for cell numbersare shown as the mean ± SEM, N = 5

The effects of treatment with the compound of formula (I) on macrophageand neutrophil accumulation in BALF following exposure to cigarettesmoke were determined (Table 6). The cigarette smoke model used for thisstudy is reported to

TABLE 6 The effect of treatment with the compound of fomula (I) ±fluticasone propionate on cigarette smoke-induced cell accumulation inmurine BALF. Treatment^(a) and dose of Cell numbers in BAL ×10⁴/mL^(b)Compound of formula (I) (% inhibition) (μg/mL) Macrophages NeutrophilsAir + Vehicle (0)  3.9 ± 0.75  2.3 ± 0.33 T + Vehicle (0) 20.1 ± 1.318.3 ± 2.2 T + (I) (20) 14.9 ± 0.65 (32) 13.0 ± 0.55 (33) T + (I) (200)10.6 ± 0.02 (59) 10.2 ± 0.83 (51) T + (I) (2000)  7.2 ± 0.40 (80)  6.5 ±0.55 (91) T + (I) (2) + FP 13.7 ± 0.63 (40) 12.4 ± 1.0 (37) T + (I)(20) + FP 10.1 ± 0.70 (62)  8.3 ± 0.72 (63) T + (I) (200) + FP  5.8 ±0.48 (90)  4.5 ± 0.49 (86) ^(a)T = tobacco smoke, FP = fluticasonepropionate dosed at 50 μg/mL; ^(b)The data for cell numbers are shown asthe mean ± SEM, N = 5.be a corticosteroid refractory system, [To, Y. et al., Am. J. Respir.Crit. Care Med., 2010, 182:897-904; Medicherla, S. et al., J. Pharmacol.Exp. Ther. 2008, 324:921-9] and the data reveal that dexamethasone(0.3-10 mg/kg, p.o.) was, as anticipated, inactive. The effects oftreatment with the compound of formula (I) on BALF neutrophils and onactivated alveolar macrophage numbers demonstrate that it possessesanti-inflammatory activity when administered as a monotherapy. Moreover,when the compound of the present disclosure was co-administered withfluticasone propionate, at a dose which lacks any significant effect asmonotherapy, a marked enhancement of anti-inflammatory activity wasdetected.

In summary, the compound of the invention is a potent inhibitor of bothPI3 kinase δ and γ isoforms. The in vitro profile translates into abroad anti-inflammatory phenotype in vivo. In this setting, theinhibitory effects of the compound disclosed herein versus PolyI:C-induced cell accumulation in the airways is notable. It is alsoparticularly striking that, unlike selective inhibitors of PI3 kinase δ,treatment with the compound disclosed herein alone results in markedinhibition of cigarette-smoke induced airways inflammation and thatthese effects occur at lower doses when it is co-administered with acorticosteroid, fluticasone propionate, under conditions where treatmentwith the corticosteroid alone is without effect.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

1. A compound of formula (I)

that is6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamideor a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof.
 2. Apharmaceutical composition comprising a compound according to claim 1,in combination with one or more pharmaceutically acceptable diluents orcarriers.
 3. A method of treatment of a condition selected from: COPD(including chronic bronchitis and emphysema), asthma, paediatric asthma,cystic fibrosis, sarcoidosis, idiopathic pulmonary fibrosis, allergicrhinitis, rhinitis, sinusitis, allergic conjunctivitis, conjunctivitis,allergic dermatitis, contact dermatitis, psoriasis, ulcerative colitis,inflamed joints secondary to rheumatoid arthritis or osteoarthritis,rheumatoid arthritis, pancreatitis, cachexia, inhibition of the growthand metastasis of tumours including non-small cell lung carcinoma,breast carcinoma, gastric carcinoma, colorectal carcinomas and malignantmelanoma which comprises administering to a subject an effective amountof a compound of formula (I) according to claim
 1. 4. A method oftreatment of a condition selected from: COPD (including chronicbronchitis and emphysema), asthma, paediatric asthma, cystic fibrosis,sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis,sinusitis, allergic conjunctivitis, conjunctivitis, allergic dermatitis,contact dermatitis, psoriasis, ulcerative colitis, inflamed jointssecondary to rheumatoid arthritis or osteoarthritis, rheumatoidarthritis, pancreatitis, cachexia, inhibition of the growth andmetastasis of tumours including non-small cell lung carcinoma, breastcarcinoma, gastric carcinoma, colorectal carcinomas and malignantmelanoma which comprises administering to a subject an effective amountof a pharmaceutical composition according to claim
 2. 5. A method oftreatment of a condition selected from: asthma, chronic bronchitis andCOPD which comprises administering to a subject an effective amount of acompound of formula (I) according to claim
 1. 6. A method of treatmentof a condition selected from: asthma, chronic bronchitis and COPD whichcomprises administering to a subject an effective amount of apharmaceutical composition according to claim
 2. 7. A method accordingto claim 5, wherein the compound of formula (I) is administeredtopically to the lungs.
 8. A method according to claim 6, wherein thepharmaceutical composition is administered topically to the lungs.
 9. Anintermediate of formula (II):

wherein LG₁ represents a leaving group; or a protected derivate thereof.